Transcriptional regulatory mechanisms are critical for normal growth control and development. In recent years, key elements of transcription have been recognized to be mediated by chromatin modification, often through large macromolecular complexes which combine modifying enzymes with the basic transcriptional machinery. One major form of chromatin modification is histone acetylation. Disruption of regulated chromatin acetylation and deacetylation has been increasingly correlated with loss of normal metabolism, growth control, and development of cancer. Recognition that the SIR2/HST gene family encodes a chromatin acetylation modifying activity places these genes even more centrally in regulatory circuits for genomic control. The SIR2/HST family of genes is perhaps the most broadly conserved family regulating chromatin and gene expression and includes members in the Archaea, the eubacteria, and in all eukaryotes examined. Progress toward understanding the mechanism of function of Sir2p and its homologues came from the recent discovery that these proteins have intrinsic NAD-dependent protein deacetylase (NAD-DAC) activity. This activity provides important insight into chromatin and chromosomal control, however, it also raises a number of questions, the answers to which are critical for understanding the biological function, specificity, and regulation of this newly defined class of enzymes. Experiments to address these questions are the foundation for the proposed research. The long-term goal of our research is to understand the in vivo regulation, targeting and specificity of the SJR2/HST family of NAD-DACs. This goal will be approached through three specific aims: 1. To define the enzymatic activity of Sir2 and the Hst proteins. 2. To establish a molecular definition of the macromolecular complexes through which the enzymes act. 3. To evaluate the functional significance of the NAD-DAC complexes and their genomic targeting. These aims will be accomplished through a combination of molecular genetic, cell biological and biochemical approaches that will be extended with emerging microarray technologies.